Hormone biosynthesis requires the selective packaging of hormones into secretory vesicles. The grant focuses on the budding of secretory vesicles from the trans Golgi network (TGN), and the roles of phospholipid metabolism and protein factor in that budding. Eventual goals include rational drug design to treat pathological conditions of hormone secretion. The basic scheme being pursued involves activation of phospholipase D in the TGN by ADP-ribosylation factor-1 (ARF-1). Then phospholipase D (PLD) in turn generates phosphatidic acid (PA). Aim 1: The role of PA accumulation in regulating the biosynthesis of phosphatidylinositol 4,5 bisphosphate (PIP2) will be examined. The hypothesis is that PA regulates PIP2, which controls budding from the TGN and also maintenance of Golgi architecture. Aim 2: Aim 1 will be extended to other cells to see what aspects of hormone secretion, Golgi morphology, constitutive secretion, and lysosomal vesicle release depends on PA production. Aim 3: Purification of protein factors, which promote vesicle budding, will be pursued using standard protein purification approaches. The hypothesis is that PLD is recruited to the TGN and activated via a protein complex consisting of ARF-1 and other proteins. Native and epitope tagged versions of ARF-1 will be used in conjunction with cross-linking agents to identify the proposed protein complex.